Lactic acid bacteria (LAB) are intensively used in the dairy industry for making different animal milk fermented products, such as e.g. yoghurt or cheese, and have achieved Generally Recognized As Safe (GRAS) status. Studies have shown that some lactic acid bacteria produce vitamin K2. The quantity of vitamin K2 synthesized by these bacteria generally varies from 29 to 90 μg/L for fermented milk (Morishita et al., 1999).
Vitamin K is important for a number of human/animal health issues such as bone health. In nature, vitamin K is present in two forms, K1 (phylloquinone) in green plants, and K2 (menaquinone, MK) in many bacteria. The MK can be further classified depending on the length of the side chain (MK-n, where n denotes the number of isoprenyl side-chain units). The lactic acid bacteria Lactococcus lactis and Leuconostoc lactis are natural producers of vitamin K2 (Morishita et al., 1999), albeit normally at a low level. The vitamin K2 is a constituent of the bacterial plasma membrane where it shuttles electrons as an essential component of the respiratory chain. A third synthetic vitamin K exists, K3 (menadione).
The major dietary source of vitamin K is K1 from vegetables and oils. The highest concentrations of K1 have been shown for green leafy vegetables—for instance spinach (Kamao et al., 2007). There are also low amounts of K1 in fish and animals. K2 is less widely distributed than K1, but high amounts can be found in livers and fermented foods, such as natto fermented with Bacillus subtilis (Sato et al., 2001). Dairy products also contain K2. In a Japanese study, the MK-4 content of cream and cheese were found to be 8 μg/100 g and 5 μg/100 g, respectively (Kamao et al., 2007). In a European study, the MK-8 content in cheese was 5-10 μg/100 g, and the MK-9 content was 10-20 μg/100 g in the same product (Shearer et al., 1996). Note that during cheese production the curd is concentrated about 10-fold thus greatly increasing the concentration of vitamin K2.
K2 has a substantially longer half-life than K1 in the body, and in a human clinical trial, intake of MK-7 had a more beneficial effect on bone health compared to K1 (Schurgers et al., 2007). Vitamin K is present in the blood and can be measured by HPLC-MS.
Vitamin K is an essential co-factor for the formation of γ-carboxyglutamic acid (Gla) residues in proteins. Gla-containing proteins are important for blood coagulation and tissue calcification through binding with calcium.
Osteocalcin (Oc) is a bone matrix protein involved in the mineralization of bone. In this process vitamin K is a co-factor. Oc is dependent on three residues of Gla, and the orientation of the Gla residues helps Oc to bind tightly to calcium ions. Research so far shows that Oc plays an important role in mineralization and remodeling of the bone tissue. Furthermore, several studies have shown that high levels of undercarboxylated osteocalcin (ucOc) is associated with lower bone mineral density and higher risk for bone fracture (Vergnaud et al., 1997).
Human studies have shown that the required daily intake should be at least 1 μg/kg body weight (Booth and Suttie, 1998). This is sufficient for efficient blood clotting but research has indicated that more vitamin K is required for strong bone health. A dose of 45 mg/day e.g. about 500-fold higher of vitamin K was beneficial for postmenopausal woman regarding bone health (Iwamoto et al., 2009). This shows that vitamin K has a wide safety range. It should be noted that vitamin K2 appears to have a higher potential regarding bone health than K1.
Osteoporosis is a metabolic bone disease, characterized by a low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. Osteoporosis is an increasing problem worldwide, and it has been estimated by World Health Organization (WHO) that in 2050, 6 million hip fractures will occur. This results in enormous loss in quality of life and cost for society.
Treating postmenopausal women with K1 and K2 as a drug for osteoporosis have shown increased bone mineral density and reduced risk of fracture (Macdonald et al., 2008; Vermeer et al., 2004). In elderly, high vitamin K levels in the serum was associated with lower fracture risk (Aliabadi et al., 2008). Thus, addition of K2 in food should be beneficial for bone health. Children would in particular benefit with an increased intake of vitamin K (van Summeren et al., 2008).
In vitro, vitamin K has been shown to have anticancer effects regarding prostate cancer cells, liver and gut tumors and some leukemia cell lines (Nimptsch et al., 2008; Shearer and Newman, 2008; Yokoyama et al., 2008). Dietary intake of K2, but not K1, has been shown to have an inverse association with prostate cancer. Vitamin K2 from dairy products has been shown to be more efficient than vitamin K2 from meat (Nimptsch et al., 2008).
Dietary intake of K2, but not K1, has been shown to reduce the risk of coronary heart disease (CHD) in older men and women (Geleijnse et al., 2004). Furthermore, in another study with 16.000 people, it was shown that MK-7, MK-8, and MK-9 from cheese and curd cheese protected against cardiovascular disease (Gast et al., 2009). Further evaluation of this data-set showed that every increase of 10 μg of K2 (but not K1) decreased the risk of cardiovascular disease by 9% (Beulens et al., 2009).
Generally speaking, the amount of vitamin K2 produced by previously known wild-type LAB is not sufficiently high to make a commercially relevant product comprising vitamin K2—e.g. a dairy product with a sufficiently high amount of vitamin K2.
Accordingly, there is a need for additional lactic acid bacteria which are able to produce increased amounts of vitamin K2—see e.g. WO2008/040793A1—and for food products and medicaments that contain vitamin K2 in sufficiently high quantities to contribute to satisfying requirements and, if necessary correcting deficiencies. Furthermore, an intrinsically produced source of vitamin K2, such as that produced by for example Lactococcus lactis is to be preferred over vitamin K added as a purified compound, as this has a more “natural appeal” and will result in a more simplified product label, i.e. a “clean label”.
Further, in WO2008/040784A1 it is described that an increased amount of produced vitamin K2 can be obtained by fermenting milk with LAB under conditions, where the LAB are not in the growth phase but in what is termed “resting cells” phase in WO2008/040784A1 (see e.g. claim 1). Essentially, this is obtained by adding a relatively large amount of LAB to the milk. It was found herein that cells grown under respiration conditions (presence of heme under agitation) have a significantly increased vitamin K2 production.
Previously, we have shown that strains having an inactivated thyA gene, encoding thymidylate synthase, produce highly increased amounts of vitamin K2 (WO2010/139690A1). Although this mutant produces increased amounts of vitamin K2 it requires specialized conditions to be propagated. It would thus be more convenient with a wild-type strain that can be produced using standard production methods.